Isolation of crystalline components from visnagan



Dec. 10, 11957 E. sMm-l ErAL 2,816,113

ISOLATION 0F CRYSTALLINE COMPONENTS' FROM VISNAGAN Filed Nov. 12. 1955'ERIC SMITH a KARL VKURT HABER ISLATION. F CRYSTALLINE COMPONENTS FROMVISNAGANA4 Eric Smith, Fairlawn, andKarlKurt Haber, Jersey'Cilty,

N. J., assignors tn S. Bl.. Peniek &..company, Interporated, New York,N. Y., `a corporation ofaDelarv'axl-e4 Application Novemberv 12, 19535Serial No. 391,594.; 4 Claims. (Cl. 260-345.2)

Our invention relates to an improvement in the isolation andidentidcationl of the components of extracts from the seeds of Ammivsnaga L., an umbelliferous plant. More particularly, the invention isconcerned with an improvement in the resolution of jthe Visnaganfraction of these extracts, andwith the isolation of crystallinesubstances from that fraction by-.an improvedmethod ef-1ehreruatosraphy- Siuse ancient tlmesthe iruitdfjthe. Plaut. Awful.'visnuga.. L.. has been used as a. falls` remedy` iu; the .treatmentuff.- variuus. ailments--suehes spasms 0f. the. vreten, fer-remaval ofealeuli. and, as a dluretie.- Reeeut.. Pharmeeplugieal.l and .elirliealtests have Confirmed. these Qld. ilse?- Tlius... the. nieder. fraetieu.Qi the fetal. estive prius', .les of Ammz' vis-naga L. which is knownaslpihellin, was .separateflilt crystalline term ansiedad t9.- be.- e.goed dilatery agent. but te Cause undesirable Side .leeetierls- Anotherknown extract. the amurehells. Visuasarl 'frestienl, is an ether-sulublelay-produst 0f Khellll. menfacture, K. Samaan has isolatedy it inl-lits.crudefor` s a.. dark, olly liquid. which. distills with; de eempesltien(Quart. I, Pharm,y Pharmacol.` 4, 1.4, 1931i; ibid, '6, 1 3, 193.3). C..I.` Cavallitu. and H-` Ruelswell detained from this oily liquid.. only.au amurulreuslslassy,Predilet which proved. app.turiruatelyV t-Wiee esattire. es. Kilellin udtlie isulatedsrabbitheert (LOrs, Chem,...15,820,1.9-50). Samaan. isolated the dark, 0i1y1iql1id.il1 about v27yield fram.; the.. seeds. and Carallite aud .Roswell state; 'that thecolorless, hard` glass` constitutes somewhat less than half ofthe crudevisnaganlproduct,

It seemed desirable to us, for the purposeoflaccurate clinical; tests ofthe4 Visnagan frac fon, to.atte\n 1 pt to isolate its components in purelcr-y v ever, we found` no, information in the. disclosn .of Samaan andof Cavallito and; Rockwell as towhow, this object can be accomplished,Samaan obtainedwthe Yishagan fraction from thelfruity of Antmi visnogqas` a greenish, Oily liquid..v C evellite.- and. Rockwell lnur .ed that.liquid by fractional precipitated .from esserti selventsand theremevelefell eenlpelleflts Capella.. Qi teacting with hydrochloricacilin ethereal solution. They thusprudused a resiueus material whichtheyeliminatesraphed. en. a silica column, usine.v ether asldevelenerand; au. ether-'ethanolI mixture. es. .ellletltlll` ,that Illettner.,they isolated. a. glassyl amsrpheus Preduet which they wereu le t0tflstallze, dlthtlllhl it heflilllepparent.- rrluleeuler Weight of,404-` We; repeated this teethud; with. a. similar lui-tial material andebteiuedtlse. salue amorphous prtldtrety It appears, therefore. that theVirussen .iteetietl isa very complex mixture of coloring matters f atonstituente. oils andpliysielesieallyastire sempurleatstlie eX- astnumbraud. nature di. wlsielll, their .reletivelatelertsls `in. the, and.belaarier iulreselutleu Prueedures wasiuulsunwn at the time ef, uuriuveutie 'lt 1s ser eralzly` recognized.. that whileelttelltetesranilyis e 0f the: .most useful methods for l the resoluties ef .,triirttflttserstree-tedfwitll peutaue7 .tractant, the` extract is concentrated anddissolved in 80% 2,8 165i i3 Eatented. Dec. 10,- 1957 ICC 2 and for theisolation and identication of `thecomponents7 the selection ofadsorbents and solvents is a diicult `problem since few simpleprinciples have been developed in the laboratory'and 'their adaptationto production scales has not-so far been successful.-

We havenow discovered thatit.ispossibletoisolate pure, crystallinecompounds from the amorphous Visnagan fraction by a columnaradsorp'tion'method" which hasfproven` successful both in the laboratoryand in production. BroadlyA speaking, we have found that crystallinecomponents are separated from the amorphous -Visnagan fraction when,after removal-of coloring matters, fatty constituents, solventsandcertain components capableoffl forming oxoniumsalts, the remaining`'amorphono rnzvrteriall is chromatographed with substantiallywater-insoluble organic solvents on an adsorbent ernployed in anA amountof not less than` about ten times they amountI of said material. We havefound that in A r the purified amorphous Visnagan fraction isresolyyed'into its crystallizable components in oneoperation.

Weprefer towash thecharged. column! with a single substantiallywater-insoluble organic solvent and then to develop and fractionally toelute. the column with mixtites st silasteutlelly Wetetiuselulsle.ergenis selveuts 'et larity. We have found that pentane-ether``ncreajsing ether content are highly selective eluentsvfSilica'gel'islthe'most: useful: adsorbent in our A The 5amorphousVisnagan fraction which we use as initial material in ourchromatographing process is obtained from a concentrated4horiz/.beneextract of'Ammi uisnaga seeds. This concentrate lisextracted with/a. waterinsoluble organic solvent, preferably" orpcntane, an the Kh'ellin and most of. the` fraction known as V ginremains in the benzene concentrateasy a' sandy vpow er. The etherextract is concentrated and the concentrate is dissolved in alcohol`aiidthel solution Or if restarle was used as. ex-

alcohol. ln either case, the alcoholic solution is then treate..v.v.ithbesie leed. eeetete te retrieve impurities After de-leading the solutionwith sodium phosphate or hydrogen` sulhde, if desired, the alcoholicsolution is concentrate'dv in vacuo, the residue is taken in absoluteother and treated with ethereal hydrogen chloride wherebycomponentscapable of forming` oxonium salts are precipitated; these hydrochloridesare'l removed by filtration. The :filtratev is washed with aqueoussodium carbonate solution, dried over sodium sulfate and concentrated toa, light syrup which amounts to 25% of the. concentrate used. This syrupwas then fractionally chromatographed.

The following examples illustratethis chro'rnatographing-.v-rqss: Y

fc'tzmple lf-Syrup lot RI-811 was mixed with I ilter cell ntheproportion, of 1:1, the mixture was dried and an yaliqtlcot amountcorresponding to 3291 grams of the syrup, was put on a silica columnweighing about thirty times as much as the syrup and previously wettedwith Y Restare.

`ellltted with `prent.aLne-,ether mixtures of increasing ether content.vThe fractions were cut from the .elluatesf' on the of Khellin.

4 TABLE I i Flow increase on isolated rabbit heart caused by crystal-(a) Ether content 10-23%: Grams oir 602` Crystalline compound RI-86020.7

(b) Ether content 23-25%:

Oil 35 Crystalline compound RI-860 5.7

(c) Ether content 25%:

Pure, crystalline compound RI-832 249 Oil 122.7

Crystalline compounds {RI 870} 22.4

(d) Ether content oil 127` Crystalline compound RI-870 214 Total 1398.5

Example 2.--Another group, lot R-811-9, was mixed with filter cel in theproportion of 1:1.5. This mixture which contained a total of 3600 gramsof syrup, was put on a silica column weighing about 15 timesas 'much asthe syrup and previously wetted with pentane.

The column was then washed with pentane; the pentane concentrate yielded60 grams of oil. The column was then developed and eluted withpentane-ether mixtures as in Example l, using the increasing ethercontents'indicated below and yielding the following products in bulk:

(a) Ether content lll-25%: Grams Oil 238 Intensely yellow colored body5.6 Crystalline compound RI860 3.5

(b) Ether content 25 Oil 374 Crystalline compound RI-832 236 Crystallinecompound RI-870 293 (c) Ether content 30-50%:

Oil 728 Crystalline compound RI-870 38 Crystalline compound RI-778 182Total 2098.1

Continued elution of the columns with pentane-ether mixtures containingamounts of ether greater than 50% yielded dark-colored and, for the mostpart, resinous materials. We were able to isolate small amounts ofKhellin and Visnagan from these resins.

These crystalline compounds were found to possess the followingproperties:

RI-860.-A crystalline compound having a melting point of 297-300 C., aspecific rotation of aryl-72 i'n ethanol, but no speciiic absorption inthe ultraviolet.

range. This compound was found to reduce the flow of Ringer solution inthe isolated rabbit heart, and it seems vto be antagonistic to theaction of Visnagin which increases the flow.

Visnagan (RI-832).-A crystalline compound having Aa melting point of83-86 C., a speciiic rotation of aD-l-lOiZ in ethanol, an ultravioletabsorption spectrum ,showing an absorption minimum at 264 ma and a maxi-Ycrystalline Visnagan possesses a vasodilatory effect on the isolatedrabbit heart which is about eight times that substances, and shows thatthe LD50 of oral Visnagan 'is Imore than ten times lower than that ofKhellin.

Table II compares the toxicities of the two' `proved to be useful forclinical investigations, since it is line Visnagan and Khellin(standard) RI-832/5 2000 Same, intravenous 590 *Control number of a lotof crystalline Visnagan. RI-870.-A crystalline substance having amelting point of 132-l34 C. and a specific rotation of D4-24 in ethanol.This substance showed an ultraviolet absorption closely similar to thatof Visnagan but dilering distinctly therefrom in its higher absorptionat lower wave lengths (Fig. 1). It proved to be extremely toxic to theisolated rabbit heart, causing stoppage of the heart in a short time.

While the ether content of the pentane-ether mixtures r employed inthese examples increased from 10 to 50%, 'we have foundthat widervariations between about 2.5 and 50% are operable` and give satisfactoryresults. In

other chromatographic runs in which the eluates were more closely cutthan in the above example, by the use of the spectrophotometric assaydescribed above, We have also encountered small amounts of additionalcrystalline fractions which we did not further identify.

We have found that instead of pentane, other hydrocarbons such ashexane, can'be employed in the solvent mixture. The syrupy initialmaterial is admixed with filter cel, preferably in the proportion of1:1, but a mixture containing 2 parts by weight of filter cel for onepart by weight of syrup has been fractionally eluted with satisfactoryresults.

Thus, we have been able to prove that the crude, amorphous Visnaganfraction which could not be resolved into its components by the knownmethods, is indeed a highly complex mixture `of amorphous constituents.Those skilled in the art know that it is diticult to resolve amorphousmixtures into their components and that is generally even more diflicultto obtain pure, crystalline compounds from such mixtures. Nevertheless,after much experimentation, we have obtained the resolution of the crudeVisnagan7 fraction and we have developed a new method of fractionalelution, as described and claimed, which has enabled us, not only toisolate individual components, but also to obtain them in pure,crystalline form in only one chromatographing operation, particularlythe pure, crystalline Visnagan which was isolated in good yields of upto about 8% and considerably less toxic than Khellin and about 8 timesmore potent as a vasodilatory agent.

What we claim is: g

l. The process of separating crystalline compounds from extracts of theseeds of Ammi vsnaga L. substantially free from coloring matters, fattyconstituents, solvents and components capable of forming oxonium saltswhich process comprises chromatographing the extracts in admixture witha filter cel on a silica gel column,

rst with pentane and then with pentane-ether mixtures of increasingether content of betwen 2.5 to about 50 percent, the silica gel beingemployed in the amount of -100 times the amount of the extract.

2. Theprocess of claim 1 in which the extracts are chromatographed withpentane-ether mixtures of increasing ether con-tent of between about 10to 30%, and the silica gel in the column has a weight about thirty timesthe weight of the extract.

3. The process of isolating crystalline compounds from extracts of theseeds of Amm vsmzga L. substantially free from coloring matters, fattyconstituents, solvents and components capable of form-ing oxonium saltswhich process comprises admixing said extracts with lter ce1 in theproportion of 1:1 by weight, putting the mixture on a silica column -inwhich the weight of the silicagel is about thirty times that of theextract, developing the column with pentane, and then fractionallyeluting it with ether-pentane mixtures, first with a mixture containingfrom 10-23% of ether, and nally with a mixture `containing from about25-35% of ether, cutting from the resulting eluates two fractions andconcentrating said fractions whereby the following products areobtained: (a) a crystalline substance melting at 83-86 C., having aspecific rotation of [a]Dl-10i2 -in ethanol, an ultraviolet absorptionspectrum showing a minimum of 264 my. and a maximum of 322 mit, amolecular weight, determined cryoscopically, of 388, and the probableformula C21H24O7; and (b) a crystalline substance melting at 132-134 C.,having a specific rotation [a]D}-24 in ethanol, an ultravioletabsorption spectrum showing a 6 minimum at 264 mp. and a maximum at 322mn and a strong end `absorption against the lower wave length range, amolecular weight determined cryoscopically, ot 386, and the probableformula C21H22O7.

4. The process of separating crystalline compounds from extracts of Ammvsnaga L. substantially free from coloring matters, fatty constituents,solvents and components capable of forming oxonium salts which processcomprises chromatographing the extracts in admixture with a filter celon a silica gel column, first with a compound selected from the groupconsisting of pentane and hexane and then with a mixture selected fromthe group consisting of pentane-ether mixtures and hexane-ethermixtures, said mixtures having an increasing ether content of between2.5 to about percent, the silica gel being employed in t-he amount of10-100 times t-he amount of the extract.

References Cited in the le of this patent UNITED STATES PATENTS2,608,560 Aschner et al Aug. 26, 1952 FOREIGN PATENTS 585,224 GreatBritain Feb. 3, 1947 OTHER REFERENCES

1. THE PROCESS OF SEPARATING CRYSTALLINE COMPOUNDS FROM EXTRACTS OF THESEEDS OF AMMI VISNAGA L SUBSTANTIALLY FREE FROM COLORING MATTERS, FATTYCONSTITUENTS, SOL-VENTS AND COMPONENTS CAPABLE OF FORMING OXONIUM SALTSWHICH PROCESS COMPRISES CHROMATOGRAPHING THE EXTRACTS IN ADMIXTURE WITHA FILTER CEL ON A SILICA GEL COLUMN. FIRST WITH PENTANE AND THEN WITHPENTANE-ETHER MIXTURESES OF INCREASING ETHER CONTENT OF BETWEEN 2.5 TOABOUT 50 PERCENT, THE SILICA GEL BEING EMPLOYED IN THE AMOUNT OF 10-100TIMES THE AMOUNT OF THE EXTRACT.